Recent evidence suggests that mammalian adipocyte turnover continues throughout adult life. While it has long been thought that adult adipocytes are generated from a stem cell population, recent lineage mapping experiments in mice provide strong evidence for an adipocyte precursor residing within adipose tissue. Many questions regarding the role of adipocyte turnover during normal homeostasis and in obesity remain incompletely elucidated, in large part due to limitations inherent to existing methods used to quantify adipogenesis, in vivo. This provided rationale to develop new quantitative methods to measure adipogenesis in vivo, based on non-toxic stable isotope tracer methodology and combined with two mass spectrometry platforms, including high resolution multi-isotope imaging mass spectrometry (MIMS). These methods enable the precise quantification of stable isotope tracers in samples ranging from pooled cell populations down to subcellular domains. Preliminary data generated with these new stable isotope approaches demonstrates a dramatic age-dependent decline in the plasticity of the white adipocyte pool, particularly in subcutaneous adipose tissue. Statistical modeling of experimental data generated with stable isotope methodology led to the novel concept that new adipocytes are generated from progenitors without a preceding division step and inform the guiding hypothesis of this study: that obesity-related metabolic diseases, such as type 2 diabetes mellitus, arise due to a failure of adipose tissue plasticity. Two inter-related specific aims emerged from this central hypothesis. Aim 1 will apply multi-isotope imaging mass spectrometry (MIMS) to test the hypothesis that new adipocytes are born from differentiating adipocyte progenitors without a self-renewing division step. This hypothesis runs counter to the current paradigm, which holds that adipocyte progenitors undergo a prerequisite clonal expansion step during adipogenic differentiation. Moreover, such a phenomenon could result in exhaustion of the progenitor pool, which may be particularly relevant at extremes of age and adiposity. Aim 2 will test the test the hypothesis tha correction of a cell cycle defect by genetic gain-of-function is sufficient to drive adipogenesis, n vivo. By generating a new platform to manipulate progenitor function in vivo, this aim will lay the foundation for future experiments testing whether augmenting progenitor-mediated refreshment of the adipocyte pool will protect against the metabolic complications of obesity and aging. This proposal views obesity through the lens of regenerative medicine, proposing the generation of new fat cells as the ideal response to the physiologic insult of caloric excess. This proposal holds promise to identify new and direct links between progenitor cell cycle activity and systemic metabolism, thereby revealing novel opportunities for targeted therapies for obesity related diabetes mellitus.